NK cells contribute to immunity against viral infection, but information on their role in defense against intracellular bacteria is limited. Innate defenses are important in the immune response to Mycobacterium tuberculosis because two-thirds of close contacts of tuberculosis patients remain healthy and have negative tuberculin skin tests. This suggests that innate immunity controls the infection before T cells recognize mycobacterial antigens and mount a delayed type hypersensitivity response. Understanding innate immunity to M. tuberculosis will enhance our capacity to develop vaccines that protect against tuberculosis, including the bioterrorist threat of multidrug-resistant tuberculosis. Recently, we found that NK cells use the NKp46 receptor to lyse M. tuberculosis infected monocytes. This proposal will characterize the mechanisms by which NK cells contribute to innate immunity against M. tuberculosis, through the following aims. 1) Identify novel ligands for NKp46 on M. tuberculosis-infected mononuclear phagocytes. NKp46 ligand(s) will be identified by immunoprecipitation, followed by mass spectrometry. The ligand will be cloned and expressed, allowing production of recombinant protein and antibodies. These tools will be used to confirm the interactions between NKp46 and its ligand(s) in vivo;2) characterize the mechanisms by which NK cells lyse infected mononuclear phagocytes. We will evaluate the mycobacterial factors and effector mechanisms that mediate lysis of infected cells such as Fas, perforin/granzyme and ATP-mediated lysis;3) Delineate the signaling pathways by which NKp46 mediates lysis of M. tuberculosis-Infected mononuclear phagocytes. We will use specific pathway inhibitors, measure enzyme phosphorylation and kinase activity, perform immunoprecipitation and Western blotting, and alter levels of individual signaling molecules through use of viral expression vectors expressing positive or dominant negative constructs;4) Characterize the NK cell subpopulations that mediate lysis of M. tuberculosis-infected mononuclear phagocytes. We will study NK cell subpopulations expressing specific patterns of cell surface markers and cytokines, using multiparameter cell sorting. These studies will provide fundamental information on the innate immune responses and lay the groundwork for development of vaccines that take advantage of the functional capacity of NK cells. These findings will facilitate development of immunomodulatory strategies to boost the immune response to M. tuberculosis, including multidrug-resistant tuberculosis.